Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that contains SH3 and SH2 domains, but lacks an SH4 domain that promotes membrane localization. Lack of membrane targeting facilitates flexibility in its intracellular localization and access to substrates. In differentiated prostate epithelial cells, PTK6 is primariy found in the nucleus. However, active PTK6 associates with the plasma membrane in prostate cancer, and its increased expression is correlated with metastasis and poor survival. We found that membrane-associated PTK6 promotes cell transformation, the epithelial mesenchymal transition, and tumor cell metastasis, while nuclear PTK6 inhibits growth. We designed a collaborative proposal to address the question, How is PTK6 recruited to the plasma membrane where it promotes tumorigenesis? Our preliminary data suggest PTK6 directly binds phosphate-rich lipids, including phosphatidylinositol-(3, 4, 5)-trisphosphate (PIP3), at the plasma membrane. Increased production of PIP3 is commonly observed in cancer due to activation of PI3K and/or mutation of the tumor suppressor lipid phosphatase PTEN. We hypothesize that phosphate-rich lipids, such as PIP3 regulate the recruitment and activation of PTK6 at the plasma membrane to drive tumorigenesis and metastases. Highly synergistic studies proposed by Dr. Tyner, a cell biologist, and Dr. Gaponenko, a structural biochemist, use an array of biochemical, biophysical, molecular, genetic and cell biology approaches to: (i) Investigate PTK6 lipid binding; (ii) Identify domains in PTK6 required for lipid binding; (iii) Determine contributions of PI3K/PTEN/PIP3 to activation of PTK6 at the membrane in prostate cancer cells; and (iv) Examine contributions of PTK6 membrane binding to prostate tumorigenesis in mice with prostate specific deletion of the gene encoding the tumor suppressor PTEN. The results of this research will define a novel mechanism for intracellular tyrosine kinase membrane targeting and activation, and could identify novel strategies to inhibit PTK6 in cancer. In additio, alterations in PTK6 intracellular localization or expression may provide a unique marker for staging prostate cancers.